The present invention relates to a process for detecting differences in protein compositions, including proteins bearing post-translational modifications, and more particularly, to a process utilizing a matched pair of labeling reagents for detecting such differences.
Researchers studying various aspects of cell biology use a variety of tools to detect and monitor differences in cell structure, function and development. An essential part of studying cells is studying the differences and similarities in the protein composition between the different cell types, stages of development and condition. Determining differences in the protein content between normal and cancerous cells or wild type and mutant cells, for example, can be a valuable source of information and a valuable diagnostic tool.
Mixtures of proteins can be separated into individual components by various means, including electrophoresis and chromatography. Separation according to differences in mass can be achieved by electrophoresing in a polyacrylamide gel under denaturing conditions. One dimensional and two dimensional gel electrophoresis have become standard tools for studying proteins. One dimensional SDS (sodium dodecyl sulfate) electrophoresis through a cylindrical or slab gel reveals only the major proteins present in a sample tested. Two dimensional polyacrylamide gel electrophoresis (2D PAGE), which separates proteins by isoelectric focusing, i.e., by charge in one dimension and by size in the second dimension, is the more sensitive method of separation and will provide resolution of most of the proteins in a sample.
The proteins migrate in one- or two-dimensional gels as bands or spots, respectively. The separated proteins are visualized by a variety of methods; by staining with a protein specific dye, by protein mediated silver precipitation, autoradiographic detection of radioactively labeled protein, and by covalent attachment of fluorescent compounds. The latter method has been heretofore only able to be performed after the isoelectric focusing step of 2D PAGE. Immediately following the electrophoresis, the resulting gel patterns may be visualized by eye, photographically or by electronic image capture, for example, by using a cooled charge-coupled device (CCD).
To compare samples of proteins from different sources, such as different cells or different stages of cell development by conventional methods, each different sample is presently run on separate lanes of a one dimensional gel or separate two dimensional gels. Comparison is by visual examination or electronic imaging, for example, by computer-aided image analysis of digitized one or two dimensional gels.
Two dimensional electrophoresis is frequently used by researchers. O""Farrell, P. H., xe2x80x9cHigh resolution two-dimensional electrophoresis of proteinsxe2x80x9d, Journal of Biological Chemistry, 250:4007-4021 (1975), separated proteins according to their respective isoelectric points in the first dimension by the now well known technique of isoelectric focusing and by molecular weight in the second dimension by discontinuous SDS electrophoresis. Garrels, J. I., xe2x80x9cTwo-dimensional Gel Electrophoresis and Computer Analysis of Proteins Synthesized By Clonal Cell Linesxe2x80x9d, Journal of Biological Chemistry, Vol. 254, No. 16, 7961-7977 (1979), used a two dimensional gel electrophoresis system to study the pattern of protein synthesis in nerve cells and glial cells. Garrels conducted a comparative analysis of data from multiple samples to correlate the presence of particular proteins with specific functions. Computerized scanning equipment was used to scan a section of the gel fluorogram, detect the spots and integrate their densities. The information was stored and plotted according to intensity in each of several different scans.
Urwin, V. E. and Jackson, P., xe2x80x9cA multiple High-resolution Mini Two-dimensional Polyacrylamide Gel Electrophoresis System: Imaging Two-dimensional Gels Using A Cooled Charge-Coupled Device After Staining With Silver Or Labeling With Fluorophorexe2x80x9d, Analytical Biochemistry 195:30-37 (1991) describes a technique wherein several isoelectric focusing (IEF) gels were used to separate proteins by charge, then loaded onto a gradient slab gel such that the IEF gels were positioned end to end along the top of the slab gel. The gels were then electrophoresed. The resulting protein spots were visualized either by staining the second dimensional slab gel with silver or by fluorescent labeling following the isoelectric focusing step. Labeling must take place after the first electrophoresis, i.e., the isoelectric focusing because the presence of the fluorescein label on the protein changes the isoelectric point of the protein when subjected to electrophoresis. In addition, the label attaches to a sulfur on the protein forming an unstable bond which would tend to break during isoelectric focusing if the label is attached prior to the electrophoresis step. An article by Santaren, J. et al., xe2x80x9cIdentification of Drosophila Wing Imaginal Disc Proteins by Two-Dimensional Gel Analysis and Microsequencingxe2x80x9d, Experimental Cell Research 206: 220-226 (1993), describes the use of high resolution two dimensional gel electrophoresis to identify proteins in Drosophila melanogaster. The dry gel was exposed to X-ray film for five days. The developed X-ray film is analyzed by a computer to determine the differences in the samples.
Two dimensional gel electrophoresis has been a powerful tool for resolving complex mixtures of proteins. The differences between the proteins, however, can be subtle. Imperfections in the gel can interfere with accurate observations. In order to minimize the imperfections, the gels provided in commercially available electrophoresis systems are prepared with exacting precision. Even with-meticulous controls, no two gels are identical. The gels may differ one from the other in pH gradients or uniformity. In addition, the electrophoresis conditions from one run to the next may be different. Computer software has been developed for automated alignment of different gels. However, all of the software packages are based on linear expansion or contraction of one or both of the dimensions on two dimensional gels. The software cannot adjust for local distortions in the gels.
Protein samples may also be separated by alternative electrophoretic or chromatography techniques. Such techniques are capable of high-resolution separation of proteins or peptides particularly in orthogonal combinations. However, current chromatographic systems tend to have lower resolving power than electrophoretic systems, ie the number of proteins or peptides capable of being separated is smaller. Typical elution traces can be found in manufacturers"" catalogues, e.g., Amersham Pharmacia Biotech xe2x80x9cBioDirectory ""99xe2x80x9d catalogue under xe2x80x9cChromatography columns and mediaxe2x80x9d starting at page 502. Nevertheless, chromatographic systems do have certain advantages over electrophoresis for some applications. For example, they are often easier to automate and it is usually easier to obtain samples of the proteins following separation.
For these reasons, separation by chromatographic systems for proteome profiling for example, is of interest. For example, Opiteck and colleagues have published examples of two-dimensional chromatographic systems where fractions eluted from a chromatographic separation system are applied to a second chromatographic system. (See specifically, Opiteck, Lewis and Jorgenson, Anal. Chem, vol. 69, 1518, (1997) which describes the use of a cation exchange system in combination with a reverse phase chromatographic system, and Opiteck et al., Anal Biochem., vol. 258, 349, (1998), which describes the use of size exclusion chromatography in combination with reverse phase chromatography.) The particularly low resolving power of size exclusion chromatography is alleviated in the latter paper by using 8 size exclusion columns in series prior to further fractionation of the eluent by reverse phase chromatography. A theoretical resolving power of 800 proteins was estimated for this system. The limited resolving power of certain chromatographic and electrophoretic systems can also be overcome at the analysis stage. Mass spectrometry is becoming widely used for protein identification following chromatographic or electrophoretic separation and can itself be used as a separation method based on mass. For example, Jensen et al., Anal. Chem. Vol. 71, 2076, (1999) describes the use of capillary isoelectric focusing as a separation method and then uses electrospray ionisation Fourier transform ion cyclotron resonance mass spectrometry to further separate proteins in the eluent from the isoelectric focusing system, as well as provide a means of identification.
The object of the present invention is to eliminate the problems associated with gel distortions or column variability and to provide a simple, relatively fast and reliable method of comparing and contrasting the protein content of different samples.
The foregoing objects have been achieved by the process of the present invention wherein differences, if any, between multiple samples of proteins, for example, those extracted from different cells or obtained from other sources, are detected by labeling each sample of such proteins with a different one of a set of matched luminescent dyes. Proteins, as used herein, includes proteins bearing post translational modifications and portions thereof, including peptides. The matched dyes have generally the same ionic and pH characteristics but absorb and/or fluoresce light at different wavelengths, producing a different color fluorescence. In addition, the dyes should be similar in size. After an incubation period sufficient to permit the formation of covalent bonds between the dye and one or more attachment sites on the proteins, the labeled samples are then mixed together and the proteins separated in a single separation process. Separation may be by electrophoresis or by chromatographic methods. When separation is by electrophoresis on a single gel, the proteins common to each sample co-migrate to the same position. Similarly, when separation is by chromatographic means in a column, for example, the proteins common to each sample migrate to the same position. Proteins which are different will migrate alone to different locations on the gel or at different times from the column and will fluoresce different colors, thereby identifying which initial sample has one or more proteins which differ from the other initial sample or samples.
The invention also includes a kit for performing the method of the present invention. The kit includes the matched set of dyes, and may also include materials for separating the proteins. Quench materials for stopping the reaction between the protein and the dye when necessary, may optionally be provided. Those materials may comprise, for example, electrophoresis gels or chromatography columns.